In Vivo Fluorescence Image Analysis System In Assessing Efficacies Of Ag85A And Ag85B DNA Vaccines In Treatment Of Bladder Cancer Cell-implanted Tumors In Mice

Objective:We are about to extract a large number of PVAX1-Ag85A and PVAX1-Ag85B plasmid,establish Bladder cancer transplanted visualization model emitted red fluorescence and evaluate the effect of respectively anti-tumor immunity treatment of Ag85A DNA vaccine and Ag85B DNA vaccine alone by technology of red fluorescent imaging in vivo and imaging features. It is to provide a basis research for the application of the optical molecular imaging of tumor and obtain intuitive and reliable theoretical basis for the development and application of DNA vaccines.Methods:Firstly, the bacteria (E.coli JM109) of plasmid which contained PVAX1-Ag85A or PVAXl-Ag85B were largely cultivated in liquid medium. Secondly, Ag85A-plasmid or Ag85B-plasmid were enormously extracted by kit.Then we carried out identification with enzyme digestion.Finally, it was quantified to satisfy concentrations (1mg/ml) in experiments and was reservation. We transferred gene which Contains Discosomasp.red fluorescent protein (DsRed) to bladder transitional cell carcinoma (BTT) cells in mouse, selected, largely cultivated and then planted in hind thigh of 615 mice. We established Bladder cancer transplanted visualization model which could emitt red fluorescence.The 24 mice's successful models were randomly divided into PVAX1-Ag85A DNA vaccine group(abbreviation:Ag85A group), PVAX1-Ag85B DNA vaccine group(abbreviation:Ag85B group) and saline group and each group has 8 mice. On the 6th,13th,20th days after planting tumor cell we respectively injected Ag85A,Ag85B and saline into the muscle of mice in the right hind limb. We observed the tumor and imaging features in fluorescence intensity about each group by intake of fluorescence imaging in vivo on the 6th,13th,20th,27th days after planting of tumor cell.On the 28th day, the mice of each group were killed to segregate liver, lung, kidney and abdominal lymph nodes.Then these tissue were cut into small sliced(lmm3)and were observed under fluorescence microscope.Results:1. PVAX1-Ag85Aplasmids and PVAX1-Ag85B plasmids were successfully established. T extracted he plasmids was less impurities and able to satisfy the experimental needs.2. BTT cells stably which were transfected with DsRed genes(BTT-DsRed) were successfully established. Transfected cells had good viability.And their red fluorescence did not weaken with passages of cell. 3. Bladder cancer transplanted models fired red fluorescent on mice were successfully established.4. In the first week and the second week after planting of tumor cells in the right hind limb, the red fluorescence imaging results in vivo showed that tumor fluorescence intensity in each group had no statistically significant difference (P> 0.05). In the third week, the red fluorescence imaging results in vivo showed that the tumor fluorescence intensities values in Ag85B DNA vaccine group were significantly lower than that in the saline group and the difference was statistically significant (P<0.05). In the fourth week, tumor fluorescence intensit values in both Ag85A and Ag85B DNA vaccine groups were significantly lower than that in the saline group and the difference was statistically significant (P<0.01). But the difference between Ag85A and Ag85B group was not statistically significant (P> 0.05).5. There were no tumor cells in Liver, lung and kidney of all mice. Distant lymph node metastasis rate in Ag85B group was significantly lower than those in the saline group (25%vs 87.5%) and Ag85A group (25%vs 62.5%). the difference was statistically significant (P<0.05).Conclusion:1. It could dynamically, objectively, sensitively and visually evaluate true effect of anti-tumor treatment on DNA vaccine by technology of red fluorescent imaging in vivo to uptake the fluorescent imaging features of transplanted tumor.2. Ag85 A or Ag85B DNA vaccine can significantly reduce fluorescence intensity for bladder transplanted tumors in mice and have tumor immune response.3. The transferred way of mice's bladder subcutaneously transplanted carcinoma was mainly lymph node metastasis.Ag85B DNA vaccine has a role in suppressing tumor distant metastasis.